Dihydrofolate reductase - thymidylate synthase (Trypanosoma brucei)

• Target (protein/gene name): Dihydrofolate reductase – thymidylate synthase
• NCBI Gene # or RefSeq#: 3658761
• Protein ID (NP or XP #) or Wolbachia#: XP_846171.1
• Organism (including strain): Trypanosoma brucei TREU927
• Etiologic Risk Group (see link below): Risk Group 2
• Background/Disease Information (sort of like the Intro to your Mini Research Write up):
  • The protozoan organism Trypanosoma brucei is responsible for the disease African Trypanosomiasis, also known as African Sleeping Sickness. The organism is considered a Risk Group 2 organism and is considered a neglected tropical disease, according to TDR Targets Database. The protist is transmitted through the bite of a tsetse fly and it is actually possible for a person to be infected with this disease yet show no sign of any symptoms until many months or years later. Without any treatment, this disease occurs in two stages: the first stage symptoms include fever, joint pain and itching and in the second stage includes infection to the central nervous system, causing changes in behavior, loss of certain motor functions and coordination. The damage to the central nervous system can affect normal sleep patterns and thus gives its name. There is currently no known drug to treat African Trypanosomiasis.
• Essentiality of this protein: After six days, this protein is essential to the organism.

1. Gene/Ortholog: Tb927.7.5480 (OG4_10927); Phenotype: no significant loss or gain of fitness in bloodstream forms (3 days); Source study: alsford
2. Gene/Ortholog: Tb927.7.5480 (OG4_10927); Phenotype: significant loss of fitness in bloodstream forms (6 days); Source study: alsford
3. Gene/Ortholog: Tb927.7.5480 (OG4_10927); Phenotype: significant gain of fitness in procyclic forms; Source study: alsford
4. Gene/Ortholog: Tb927.7.5480 (OG4_10927); Phenotype: significant gain of fitness in differentiation of procyclic to bloodstream forms; Source study:alsford
   • alsford: High-throughput phenotyping using parallel sequencing of RNA interference targets in the African trypanosome. Genome Res 2011, 21:915-924

• Complex of proteins?: Yes
• Druggable Target: 1
• EC#: 1.5.1.3
• Link to BRENDA EC# page: http://www.brenda-enzymes.org/php/result_flat.php4?ecno=1.5.1.3
  • http://www.brenda-enzymes.org/literature/lit.php4?e=1.5.1.3&r=392285
    • Link to a PubMed article that studied DHFR-TS gene isolation and expression

Fig. 1: BRENDA Enzyme mechanism schematic for the reaction catalyzed by DHFR

• Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
  • link to Sigma: http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Enzyme_Assay/dihydrofolatereductase.pdf
  • link (or citation) to paper that contains assay information: http://www.ncbi.nlm.nih.gov/pubmed/11325838
  • List cost and quantity of substrate reagents and supplier
    • A. 50 mM Potassium Phosphate Buffer, pH 6.5 at 25°C (Sigma Prod. No. P-5379) $19.90 for 100g
      
      B. 0.11 mM ß-Nicotinamide Adenine Dinucleotide Phosphate, Reduced Form Solution
      (ß-NADPH) (Sigma Stock No. N9660)
      Pricing is for NADPH, reduced tetra (cyclohezylammonium) salt Sigma Stock No. N5130:$99.90 for 25mg
      
      C. 2.3 mM Dihydrofolic Acid Solution (DHFA) (Sigma Prod. No. D-7006)
      Price: $71.60 for 10mg
      
      D. 0.1% (w/v) Bovine Serum Albumin Solution (BSA)
      (Sigma Prod. No. A-4503) Price: $73.60 for 10g
      
      E. Dihydrofolate Reductase Enzyme Solution
      Dihydrofolate Reductase (Human) Sigma No. D6566: $241.50 for .25 units
      
      
• Structure Available (PDB or Homology model)
  • PDB #: 3RG9

• Current Inhibitors: 7,8-dihydrofolate + NADPH
• Expression Information (has it been expressed in bacterial cells): Information is shown in the experimental procedure section in link below.
  http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2958.2008.06305.x/pdf
• Purification Method: Information is shown in the experimental procedure section in link below.
  http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2958.2008.06305.x/pdf
• Image of protein (PyMol with features delineated and shown separately):

Fig 2: PyMol cartoon representation of the protein 3RG9.

• Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):

MLSLTRILRKKIPVHELAGKISRPPLRPFSVVVASDEKGGIGDGGTIPWEIPEDMQYFRR
VTTNLRGKNVKPSPSKRNAVVMGRKTWDSLPPKFRPLSNRLNVVLSRSATKEQLLAGIPD
PIKRAEAANDVVAVNGGLEDALRMLVSKEHTSSIETVFCIGGGTIYKQALCAPCVNVLQA
IHRTVVRPASNSCSVFFDIPAAGTKTPEGLELVRESITDERVSTGAGGKKYQFEKLVPRN
SEEEQYLNLVGRIIDEGCTKCDRTGVGTRSLFGAQMRFSLRNNRLPLLTTKRVFWRGVCE
ELLWFLRGETNAKLLSDKGIHIWDGNGSRAFLDSRGLTDYDEMDLGPVYGFQWRHFGADY
ISCKVDSEGKGVDQIANIVKSLIENPDDRRMICTAWNPAALPRMALPPCHMMAQFYVSNG
ELSCMLYQRSCDMGLGVPFNIASYALLTFLMAKASGLRPGELVHTLGDAHVYSNHVEPCR
KQLKRVPRXFPFIVFKQDKEFLEDFQESDIEVIDYSPYPVISMEMAV

• Length of your protein in Amino Acids: 527
• Molecular Weight of your protein in kiloDaltons using the Expasy ProtParamwebsite: 58.8 kDa
• Molar Extinction coefficient of your protein at 280 nm wavelength: 58620 M-1 cm-1
• TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.

Figure 3: TMpred output determining the probability of the appearance of 3RG9 (PDB ID) as a membrane bound protein

TMpred indicates that it is possible that there are two transmembrane helices.

• CDS Gene Sequence (paste as text only): TCCGTCCACTGTCTAACCGCCTTAATGTTGTGCTGTCACGAAGTGCGACAAAGGAACAGTTGTTGGCTGG
  TATCCCTGATCCCATCAAACGAGCGGAGGCTGCGAATGATGTGGTTGCTGTGAACGGTGGGCTTGAAGAC
  GCACTTCGGATGTTGGTAAGTAAGGAGCACACATCTTCTATCGAAACGGTGTTTTGCATTGGTGGCGGGA
  CTATATATAAACAAGCCCTTTGTGCCCCATGCGTTAATGTGTTGCAAGCTATCCACCGGACGGTGGTCCG
  ACCAGCCTCAAATTCGTGTAGCGTTTTCTTTGACATCCCAGCCGCAGGGACAAAGACCCTGGAAGGACTT
  GAGTTGGTGCGGGAAAGCATAACGGACGAACGGGTATCAACCGGAGCTGGCGGAAAGAAATATCAGTTTG
  AGAAGCTTGTCCCCCGAAACAGCGAAGAGGAGCAGTATTTAAATCTCGTTGGCCGCATAATTGATGAGGG
  ATGTACGAAATGTGACCGCACGGGTGTTGGTACCCGCTCCCTCTTTGGGGCCCAGATGCGTTTCTCTCTC
  CGCAACAATCGCCTGCCATTACTAACCACGAAGCGTGTCTTTTGGCGTGGAGTATGTGAAGAGCTGTTGT
  GGTTTCTTCGGGGAGAAACAAATGCAAAGTTGCTGAGCGACAAGGGGATTCACATATGGGATGGGAATGG
  CTCACGAGCATTCCTTGACAGTCGTGGCTTGACTGATTATGATGAGATGGACCTCGGACCTGTTTATGGC
  TTCCAGTGGAGGCATTTTGGTGCCGACTATATTTCTTGCAAGGAGGATTCCGAGGGGAAAGGTGTTGATC
  AGATTGCAAACATTGTGAAGTCATTGATAGAGAACCCGGATGACCGTCGCATGATTTGTACAGCTTGGAA
  TCCGGCCGCCTTGCCACGCATGGCGCTCCCACCATGCCACATGATGGCGCAATTCTACGTGAGCAATGGG
  GAACTCTCATGCATGTTGTACCAGCGGTCATGTGATATGGGTCTTGGCGTCCCCTTCAATATTGCTTCGT
  ATGCACTGCTTACATTCCTCATGGCAAAAGCTTCTGGACTTCGGCCGGGCGAATTGGTGCACACCCTTGG
  CGATGCACATGTTTACAGTAACCATGTGGAGCCGTGTCGGAAGCAATTGAAACGCGTGCCCCGCCCGTTC
  CCTTTCATCGTGTTCAAACAAGACAAAGAGTTCCTGGAAGACTTTCAGGAAAGCGATATAGAGGTCATCG
  ATTATTCTCCGTACCCCGTAATTTCTATGGAGATGGCGGTGTAG
• GC% Content for gene: 50.88%
• CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
• GC% Content for gene (codon optimized):

Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.


References
TDR Targets Page: http://tdrtargets.org/targets/view?gene_id=13461
Pubmed - T. cruzi DHFR-TS: http://www.sciencedirect.com/science/article/pii/0166685194900760
Pubmed - T. brucei DHFR-TS: http://www.sciencedirect.com/science/article/pii/016668519500059A
Pubmed - T. cruzi DHFR-TS: http://www.sciencedirect.com/science/article/pii/S096808961000324X